JPH04330234A - Cucumber mosaic virus resistant tomato and method for preparing the same - Google Patents

Abstract

PURPOSE:To obtain a tomato having resistance to cucumber mosaic virus. CONSTITUTION:Tomato is transformed by a plasmid containing cDNA of satellite RNA to produce a cucumber mosaic virus resistant tomato.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a tomato exhibiting resistance to infection with cucumber mosaic virus and a method for producing the same.

0002

BACKGROUND OF THE INVENTION Cucumber mosaic virus is an important pathogenic virus that infects a wide range of plants. The damage to tomatoes is particularly severe, and the development of resistant varieties is desired. However, because there are no resistant strains of tomato cultivated varieties or their related wild varieties, the main focus of cucumber mosaic virus control currently relies solely on cultivation methods and indirect means using pesticides. .

[0003] In recent years, with the development of genetic recombination techniques,
It has become possible to acquire a completely new type of virus resistance through transformation that introduces viral coat protein and satellite RNA genes. It has been known that if a plant is inoculated with a weakly pathogenic virus in advance, the plant will be protected from infection by the highly virulent virus even if it is later infected with a highly virulent strain of the same virus. This effect is thought to be due to the earlier virus preventing the shedding of the coat protein that protects the genes of the later virus. Therefore, if you extract the gene for the viral coat protein and introduce it into plants using the transformation method to create a plant that produces the viral coat protein, the plant will exhibit virus resistance similar to interference. It is known that this happens. For example, tobacco mosaic virus (Powell et al., Science 232
:738-743, 1986), alfalfa mosaic virus (Tumer et al., EMBO J 6:118)
1-1188, 1987 and Roche et al., EMBO
J 6:1845-1851, 1987), potato virus X (Hemenway et al., EMBO J 7:12
73-1280, 1988), potato virus Y (
Lawson et al., Bio/Technology 8:12
7-134, 1990) and cucumber mosaic virus (Kuzo et al., Bio/Technology 6:5
49-558, 1988) has been reported to be effective in alleviating disease symptoms.

[0004] Cucumber mosaic virus is an RNA virus. This virus particle usually contains four types of RNA (
RNA1, RNA2, RNA3, RNA4), and RNA1 and RNA2 have genes related to RNA virus replication. The gene on the 5' side of RNA3 is thought to be involved in the intercellular movement of the virus. Furthermore, RNA4 is the same as the 3' side gene of RNA3, and contains the virus coat protein gene. However, some isolates of cucumber mosaic virus contain extra RNA called satellite RNA. This satellite RNA is 330-3
It is a small RNA consisting of 90 bp, and the RNA itself does not have the ability to proliferate and relies on the parent virus for its replication. An important characteristic of this satellite RNA is that it changes the disease symptoms of diseased plants. That is, when cucumber mosaic virus and satellite RNA simultaneously proliferate in host plant cells, the proliferation and accumulation of the virus is generally suppressed, and the disease symptoms of virus-infected plants are often reduced compared to plants that do not have satellite RNA. Therefore, attempts were made to suppress the proliferation of cucumber mosaic virus and the development of disease symptoms by introducing this satellite RNA into plants using an appropriate transformation method and expressing it in the plants. It has been reported to be effective (Harrison et al., Nature 328:
799-802, 1987, Agricultural, Genetex, Company, Limited JP-PAT
64-27474). In addition, attempts were made to introduce the satellite RNA of cucumber mosaic virus into tomatoes, and tomatoes with satellite RNA introduced were also produced.
The disease symptoms were amplified compared to normal ones, and practical resistance was not achieved (McGarvey et al., Biochemical
al and Biophysical Research
ch Communications 170:548
-555, 1990, Touche et al., C. R. Aca
d. Sci. Paris311, Serie
III:377-384, 1990).

[0005]

[Problems to be Solved by the Invention] As mentioned above, it has not been possible to introduce the satellite RNA of cucumber mosaic virus into tomatoes to produce tomatoes that exhibit a practically sufficient level of resistance. An object of the present invention is to provide a tomato cultivar with excellent resistance to cucumber mosaic virus.

[0006]

[Means for Solving the Problems] As a result of intensive research on the satellite RNA of cucumber mosaic virus, the present inventors found that it has no adverse effects on tomatoes such as yellowing or intensification of disease symptoms, and has an extremely high effect of suppressing disease symptoms. They succeeded in isolating satellite RNA of cucumber mosaic virus, and discovered that by introducing this satellite RNA into tomatoes, tomatoes with high resistance to cucumber mosaic virus could be produced, and this invention was completed. That is, the present invention relates to cucumber mosaic virus satellite R.
A cucumber mosaic virus-resistant tomato containing a cDNA of NA is provided.

[0007] In the present invention, it is necessary to use cucumber mosaic virus satellite RNA introduced into tomatoes that has a high effect of suppressing disease symptoms and has little adverse effects on tomatoes. The present inventors collected plants infected with cucumber mosaic virus showing slight disease symptoms, and isolated four strains of cucumber mosaic virus satellite RNA from these plants. These satellite RNAs are each S19-sa
tRNA, T43-satRNA, T62-sat
The RNA was named T73-satRNA. When tomatoes were inoculated with the satellite RNA of these cucumber mosaic viruses, T73-satRNA was found to be particularly effective in reducing disease symptoms (Masta et al., An.
n. Phytopath. Soc. Japan
56:207-212, 1990). In addition, T73-
The base sequence of satRNA is shown in FIG.
The base sequence of the satellite RNA of the strain is also described in the above-mentioned document by Master et al. However, the satellite RNA of cucumber mosaic virus used in the present invention
is not limited to these. That is, plants showing disease symptoms after being infected with cucumber mosaic virus, such as tobacco, are infected with cucumber mosaic virus, satellite RNA is extracted from plants with reduced disease symptoms by a conventional method, and this is used to infect tomatoes. By selecting those that provide excellent resistance to mosaic viruses,
It is possible to obtain satellite RNA suitable for use in the present invention.

Next, the process of producing cucumber mosaic virus-resistant tomatoes of the present invention will be explained. In the present invention, in order to introduce satellite RNA into a plant and express it, it needs to be DNA. Therefore, cDNA containing the entire length of the isolated satellite RNA can be cloned by a conventional method using synthetic primers and reverse transcriptase.

Satellite RN obtained as above
A cDNA can be introduced into tomatoes using a transformation method. Conventionally, the efficiency of tomato transformation has been low, and depending on the variety, transformation has been extremely difficult. The present inventor has developed a novel tomato transformation method that is extremely efficient and does not cause any harm to tomatoes, and has filed a patent application (Japanese Patent Application No. 2-411681). Cucumber mosaic virus can be introduced into tomatoes by this method.

[0010] In this method, Agrobacterium
Ti plasmid pTi of M. tumefaciens
Agrobacterium into which a plasmid containing a DNA region derived from the virulence region of Bo542 and cDNA of cucumber mosaic virus satellite RNA were introduced.
Transform tomatoes with M tumefaciens. Here, the DNA derived from the virulence region of pTiBo542 preferably contains virB, virG, and virC genes. A preferred example of such a plasmid is plasmid pTOK162, which will be described in detail in Examples below. The cucumber mosaic virus satellite RNA is inserted into the T region of the plasmid. This insertion can also be carried out by a conventional method using restriction enzymes, but in the case of a large plasmid with many restriction enzyme sites, such as the plasmid pTOK162, it is not possible to insert the desired DNA into the T region using the usual subcloning method. It may not always be easy to introduce it within the organization. In such cases, Agroba
cterium tumefaciens intracellular i
Homologous recombination in the n vivo system (Herrera-Esterera et al., EMBO J. 2:987-995, 1983;
Hoch et al., Science, 223:496-498
, 1984) to extract the desired DNA.
can be introduced into pTOK162. That is, for example, first, pTOK162 is
cterium tumefaciens, and a plasmid called pBR322 (containing similar plasmids) into which cDNA of cucumber mosaic virus satellite RNA is introduced. Since the DNA of pTOK162 has a portion homologous to pBR322, the pBR322 derivative will be integrated into pTOK162 by recombination via the homologous sequence. pBR322 differs from pTOK162 in that A
grobacterium tumefaciens
Agrobacterium tumefacien cannot be replicated in this state unless it is in this integrated state (called pTOK162::pBR322).
cannot survive in s. And pTOK1
pTOK162 and pBR322 derivatives can be selected based on their specific characteristics (drug resistance, etc.).
::Agrobacterium with pBR322 derivative
rium tumefaciens can be obtained. Next, Agrobac into which this plasmid was introduced
cDNA was obtained by co-cultivating Terium tumefaciens and tomato cotyledon fragments in liquid medium.
can be introduced to tomatoes. After washing the cotyledon fragments,
For example, if the plasmid has drug resistance, a transformant can be obtained by implanting the plasmid in a tomato foliage differentiation medium containing an antibiotic, and by implanting foliage showing resistance to the antibiotic in a rooting medium. For the transformants that have emerged, tomatoes resistant to cucumber mosaic virus can be obtained by examining their morphology and traits, introducing cDNA of satellite RNA, confirming mRNA transcription, and inoculating them with cucumber mosaic virus.

[0011]

[Effects of the Invention] The present invention provides a tomato that is virtually fully resistant to cucumber mosaic virus and a method for producing the same, which does not cause damage such as delayed growth or reduced fruit yield due to infection with cucumber mosaic virus. . The tomato of the present invention is a resistant tomato that shows almost no disease symptoms even when infected with the cucumber mosaic virus normal strain (O strain).

0012

[Examples] The present invention will be explained in more detail with reference to Examples below, but the Examples of the present invention are not limited thereto. In the following examples, unless otherwise specified, T. maniatis (Molecular
Cloning, Cold Spring Harb
(1982)). (1) Isolation of T73-satRNA and cloning of cDNA Various cucumber mosaic viruses were collected mainly from tobacco-producing areas in Tochigi Prefecture, and satellite RNAs of four strains of cucumber mosaic viruses were isolated. These four satellite RNA lines are each S19-sa
tRNA, T43-satRNA, T62-satR
NA, named T73-satRNA. When these cucumber mosaic virus satellite RNAs were inoculated into tomatoes, it was found that T73-satRNA was particularly effective in alleviating disease symptoms in tomatoes (Masta et al., supra). Next, cDNA containing the full length of T73-satRNA was cloned using synthetic primers and reverse transcriptase. Specifically, this operation was performed as follows. T73
- Using a synthetic probe homologous to the 3' end region of satRNA as a primer, first strand cDNA synthesis is performed, and RNA
- A DNA hybrid molecule was obtained. This hybrid molecule was converted into RNaseH.
After treatment, a second strand of DNA was obtained using a synthetic probe homologous to the 3' end of the DNA as a primer. The full-length cDNA thus obtained was transformed into plasmid pUC119.
(manufactured by Takara Shuzo Co., Ltd.) into the SmaI site and pU
C119-73sat was obtained.

(2) Introduction of satellite RNA into plasmid pTOK162 The cDNA obtained in (1) was transformed into a plant expression vector pL.
Incorporated into GV2382 by a conventional method, pLGV2382
-73 sat was obtained. This vector is digested with restriction enzyme Hind.
III and PvuII, and a DNA fragment containing the Nos promoter, T73-SatRNA cDNA, and Nos terminator was recovered. This DNA fragment was made blunt-ended with E. coli DNA polymerase I Klenow fragment. Next, pBR322 containing the spectinomycin resistance gene derived from the 2.5 kb restriction enzyme ClaI fragment of transposon Tn7 was digested with the restriction enzyme ClaI, blunt-ended with E. coli DNA polymerase I Klenow fragment, and the previous DNA fragment and T4 ligase were cut. After ligation, pYS134 containing the T73-SatRNA cDNA and the spectinomycin resistance gene was obtained. The thus obtained pYS134 was transformed into a plasmid pTOK1 with kanamycin and tetracycline resistance.
62 (Komali, Plant Cell Report
9:303-306, 1990)
bacterium tumefaciens strain LBA4404 (Hockman et al., Nature 30
3:179-180, 1983) using the three-line cross method (Ditta et al., Proc. Natl. Acad. Sci.
SA 77:7347-73511980). Next, colonies showing resistance to both kanamycin and spectinomycin were selected. The obtained resistant colonies contained two types of plasmids, pYS134 and pTOK162.
It emerged through recombination through homologous regions within. As a result, T73-s was added to plasmid pTOK162.
A plasmid incorporating the atRNA gene was constructed and named pYS143. Figure 1 shows plasmid pYS
A diagram of the construction of 143 is shown.

(3) Transformation of tomato Tomato was transformed using LBA4404 (pYS143) obtained in (2). For transformation, tomato cultivars Momotaro (Takii Seed Co., Ltd.) and JTM-7 (grown at Japan Tobacco Co., Ltd., Plant Development Research Institute) were used. After sterilizing these seeds with ethyl alcohol and sodium subzinc oxide, Linsmaier and
The cells were plated on a medium containing Skoog (1965) inorganic salts, 30 g/l sucrose and agar (0.9%). Ten days after sowing, the aseptically germinated tomato cotyledons were cut into pieces of 0.7 x 0.7 cm. 1g of this cotyledon fragment and Agrobacterium tume
Linsmaie with about 108 cells
r and Skoog inorganic salts and 30g/l
The cells were co-cultured for 40 hours in a liquid medium consisting of glucose. The cotyledon fragments were then washed with sterile water to remove bacteria, and then treated with Linsmaier and Skoog's inorganic salts, indoleacetic acid 0.3 mg/l, isopentenyl adenine 10 mg/l, kanamycin 100 m
g/l, 250 mg/l of cefotaxime, and 30 g/l of glucose. After 20 days of culture, kanamycin-resistant callus appeared from the implanted cotyledon fragments. These calli were cultured on the above medium for an additional 15 days, and as a result, stems and leaves showing kanamycin resistance appeared. Cut these stems and leaves, Lin
Inorganic salts of Smaier and Skoog, 30
g/l sucrose and cefotaxime 250 mg
The cells were implanted on an agar (0.9%) medium containing /l. After 30 days of culture, the rooted plants were cultivated in a closed room at room temperature. As a result, four regenerated plants (Sa
t-71, Sat-72, Sat-73, Sat-74
) were obtained, and for Momotaro, three regenerated plants (Sa
tm-11, Satm-12, Satm-13) were obtained.

(4) Morphology of regenerated plants A comparative investigation was conducted between the regenerated plants obtained in (3) and their parent varieties JTM-7 or Momotaro. As a result, the morphology and growth characteristics of these transformants were the same as those of the parent variety, the fertility was normal, and the fruit formation was also normal like the parent variety.

(5) T73-sat by Southern analysis
Among the regenerated plants confirmed to have introduced the RNA gene, Sat-71, Sat-72,
Satm-11, Satm-12, Satm-1
3, the introduction of the T73-satRNA gene was confirmed by Southern analysis. First, from the leaves of these five individuals, the method of Komari et al. (Komali et al., Theor Appl.
Genet 77:547-552, 1989), total DNA was extracted. The obtained total DNA was digested with the restriction enzyme Bam H1, and DNA fragments were separated by agarose gel electrophoresis. Next, these DNA fragments were transferred to a nylon membrane, Gene Screen Plus (manufactured by Jupon), and the plasmid pUC1
19-73sat Hind III, EcoRI
Southern analysis was performed on the 0.4 kb fragment using a 32P-labeled probe using the random prime method (Boehringer, manufactured by Mannheim). The results are shown in FIG. As a result of Southern analysis, a 5.8 kb band was detected in Sat-71 and Sat-72, which approximately matched the size of the Bam H1 fragment containing the introduced T73-satRNA gene. Also used as a control was JTM.
No such band was detected for -7. From this result, we found that cD of T73-SatRNA was present in these two individuals.
It became clear that NA was incorporated. on the other hand,
A similar analysis was performed on three Momotaro transformants, and as a result, introduction of T73-SatRNA cDNA was confirmed.

(6) T73-sa by Northern analysis
Confirmation of transcription of tRNA gene mRNA of T73-satRNA gene by Northern analysis of redifferentiated plants Sat-71 and Sat-72
A transcription was confirmed. First, total RNA was extracted from the leaves of these two individuals using the Thomas method (Thomas, Proc. Natl. Acad.
Sci. USA 77:5201-5205 (1980
)) with glyoxal and dimethyl sulfoxide.
After NA was denatured, it was separated by agarose gel electrophoresis. Next, these RNAs were transferred to a nylon membrane and GeneScreen Plus (manufactured by Jupon), and Northern analysis was performed using the same 0.4 kb DNA fragment as in Southern analysis as a probe. The results are shown in FIG. As a result of Northern analysis, transcription of 1.3 kb mRNA was observed in Sat-71 and Sat-72. This is the m transcribed from the predicted T73-satRNA gene.
The size corresponds to that of RNA. Further, such transcription of mRNA was not observed in JTM-7 used as a control. This result shows that T7 in these two individuals
3-SatRNA gene mRNA is transcribed. Regenerated plant Satm-11, Sa
Similar analysis of tm-12 and Satm-13 confirmed transcription of T73-satRNA gene mRNA.

(7) Expression of disease symptoms by artificial inoculation with cucumber mosaic virus Transformants Sat-71 and Sat- in which introduction of T73-satRNA cDNA and transcription of mRNA were observed
72 were inoculated with cucumber mosaic virus common strain (O strain). As a control, the parent variety JTM-
7 was used. As a result, Sat-71 and Sat-72 inoculated with cucumber mosaic virus O strain showed slight mosaic-like disease symptoms, but the morphology and growth condition were
There was no difference from the control JTM-7 which was not inoculated with cucumber mosaic virus O strain. In addition, normal fruit formation was observed as in the control JTM-7. On the other hand, JTM-7 inoculated with cucumber mosaic virus O strain had extremely delayed growth and did not form any fruit. From the above results, it was revealed that individuals in which expression of T73-satRNA was confirmed showed very slight disease symptoms even if infected with the common cucumber mosaic virus. Next, RNA extracted from Sat-71 and Sat-72 inoculated with cucumber mosaic virus O strain was analyzed by agarose gel electrophoresis. The results are shown in FIG. In addition to RNA of cucumber mosaic virus O strain, it was confirmed that T73-satRNA was proliferating. On the other hand, T73-satRNA was not observed in JTM-7, which was used as a control and inoculated with cucumber mosaic virus O strain. From this result, it is considered that the proliferation of T73-satRNA observed here brings about the effect of alleviating disease symptoms. Transformants Satm-11, Satm-12, Satm-
13 was similarly inoculated with cucumber mosaic virus O strain, but almost no disease symptoms appeared, and proliferation of T73-satRNA was observed in these transformants. From the above results, the cDNA of T73-satRNA
It has become clear that tomatoes introduced with Cucumber Mosaic Virus show only very slight disease symptoms even when infected, and are effectively resistant to the virus. Therefore, the present invention has made it possible to produce tomatoes resistant to cucumber mosaic virus, which has not been possible until now.

[Brief explanation of drawings]

FIG. 1: Diagram showing the construction of plasmid pYS143.

FIG. 2 is a diagram showing the entire base sequence of T73-satRNA.

FIG. 3 is a diagram showing the results of Southern analysis using transformants Sat-71 and Sat-72.

FIG. 4 is a diagram showing the results of Northern analysis using transformants Sat-71 and Sat-72.

FIG. 5 is a diagram showing the results of agarose gel electrophoresis of RNA of CMV-inoculated transformant Sat-71.

Claims (5)

[Claims] 1. A cucumber mosaic virus-resistant tomato comprising a cDNA of a satellite RNA of cucumber mosaic virus. 2. Claim 1, wherein the cDNA is T73-satRNA cDNA of cucumber mosaic virus.
The described cucumber mosaic virus resistant tomato. [Claim 3] Agrobacterium tum
efaciens Ti plasmid pTiBo542
A plasmid containing a DNA region derived from the virulence region of C. and a satellite RNA of cucumber mosaic virus.
Agrobacterium tum introduced with DNA
A method for tomato transformation comprising transforming tomatoes with P. efaciens. 4. The DNA derived from the virulence region of pTiBo542 contains virB, virG, and virC.
4. The method according to claim 3, comprising a gene. 5. The Ti plasmid pTiBo542
4. The method according to claim 3, wherein the plasmid containing DNA derived from the virulence region of is plasmid pTOK162.